1. Field of the Invention
The present invention relates to compounds that act, at physiologically-compatible levels, to impede the formation of amyloid in brain, including Alzheimer senile amyloid plaques in vivo. More specifically, the present invention relates to compounds that impede the transformation of dense microspheres (DMS) into amyloid. The invention also relates to methdology for the screening of compounds that possess this activity, and to treating cerebral amyloidosis by administering such compounds therapeutically.
2. Description of Related Art
There is no effective therapy for cerebral amyloidosis, which almost invariably has a fatal outcome following the onset of amyloid deposits. For example, Alzheimer""s disease is estimated to be the fourth or fifth leading cause of death in North Americans.
A universally accepted indicator of cerebral amyloidosis is the accumulation of lesions, so-called xe2x80x9csenile plaques,xe2x80x9d that are comprised in large part of amyloid fibrils. Senile plaques are spherical, ranging from 10 to 200 xcexcm in diameter, and are found occasionally in aged cerebral cortex (see below) but in larger numbers in Alzheimer-affected cortex.
Most specialists agree that reproduction of amyloid fibrils experimentally from precursor materials which are extracted, activated, or otherwise derived from human brain constitutes the best available evidence linking an agent or precursor to the progression of cerebral amyloidosis. Indeed, the literature has documented the use of such materials from human brain, normal and Alzheimer-affected, that are not already amyloid, and their transformation into amyloid.
Accordingly, a reliable indicator is available for compounds that might be effective in treating cerebral amyloidosis. In particular, it is possible to determine whether a given compound prevents the structural tansition of a brain-localized precursor to a xcex2-pleated sheet conformation and thus prevent conversion to cerebral amyloid (i.e., that displays xe2x80x9canti-amyloid activityxe2x80x9d) at physiologically acceptable levels of the active agent. See U.S. Pat. No. 5,231,170, the contents of which are incorporated by reference.
In a majority of cases, subjects with cerebral amyloidosis, including Alzheimer""s patients, display symptoms on a quantitative basis. Blessed et al., Brit J. Psychiatry 114: 797-811 (1968); Tomlinson et al., J. Neurol. Sci. 7: 331-56 (1968); Tomlinson, B. E., et al., J. Neurol. Sci. 11: 205-42 (1970); Corsellis, MENTAL ILLNESS AND THE AGEING BRAIN, Oxford University Press, London (1962); Corsellis, xe2x80x9cAgeing and the Dementias,xe2x80x9d in GREENFIELD""S NEUROPATHOLOGY 796-848, Edward Arnold, London (1976). Elderly subjects who have a small number of senile plaques are asymptomatic and are categorized by some experts as preclinical, by others as presymptomatic, and by still other authorities as normal variants. In any event, the elderly brain apparently can have a low number of senile plaques and still be categorized as xe2x80x9cnormal.xe2x80x9d When the amyloid plaque number is high, however, symptoms of dementia appear. Thus, treatment regimens that prevent the formation of amyloid plaques or that reduce the number or rate of formation of amyloid plaques are useful.
A microscopic structure, the so-called xe2x80x9cdense microspherexe2x80x9d (DMS), is found in normal brain and in brain affected by Alzheimer""s disease. See Averback, Acta Neuropathol. 61: 148-52 (1983); results confirmed by Hara, J. Neuropath. Exp. Neurol. (1986). Evidence for the existence of dense microspheres comes from microscopic histological section studies of fixed whole brain tissue, where the dense microspheres are seen to have a proteinaceous central region (xe2x80x9cDMS proteinxe2x80x9d) surrounded by continuous membrane (xe2x80x9cDMS membranexe2x80x9d).
The extraction, purification, and characterization of isolated samples of DMS and the use of DMS material have been documented. See, for example, U.S. Pat. Nos. 4,919,915 and 4,816,416, the respective contents of which are incorporated by reference.
DMS disruption is believed to commence after individual DMS reach a threshold size in the elderly or Alzheimer group. Even DMS that have reached the threshold size are quite small, with diameters on the order of approximately 10 microns or less. When the DMS disrupt, constituent protein matter transforms and redistributes to occupy a tissue volume, (anywhere between 10 to 1,000 times larger than the precedent DMS), which comprises a much larger injury focus.
Preventing the disruption of DMS would prevent the formation of amyloid plaques. If DMS disruption can be curtailed, for example, by around 25% or more, cerebral amyloid formation, and its rate of formation, induced by DMS disruption also can be reduced or prevented. Thus, reducing the size of the injury foci associated with disrupted DMS would impede the formation of amyloid plaques, by reducing the number of DMS disruptions that may be caused by an initial disruption. In addition, reducing the persistence of injury and inflammation associated with DMS disruption, including, for example, increasing the digestion and removal of DMS material through the reticuloendothelial system, would prevent the formation of amyloid plaques or, at least, reduce the number and/or rate of formation of other, secondary DMS disruptions.
Accordingly, a need exists for an approach to preventing DMS disruption. A need also exists for a method to reduce the size of the tissue volume associated with disrupted DMS (injury foci). Furthermore, there is a need for a technique to reduce the persistence of inflammatory reaction brought about by DMS disruption, as well as for methods to increase the digestion and removal of DMS material through the reticuloendothelial system.
It therefore is an object of the present invention to provide a composition and a method that are useful in impeding DMS disruption and, hence, in treating cerebral amyloidosis, a condition characterized by the presence of abnormal amounts of amyloid xcex2-protein associated plaques (senile plaques) and other amyloid deposits. It is an additional object to provide a screening approach for identifying compounds that are useful in impeding DMS disruption.
It is also an object of the present invention to provide a treatment for cerebral amyloidosis by the administration of a compound selected from a class of pharmaceutically active agents that have in common an ability to impede the disruption of intact DMS.
In accomplishing the foregoing objects, a method has been provided, in accordance with one aspect of the present invention, for treating cerebral amyloidosis, comprising the step of administering to a subject in need thereof, a pharmaceutically effective amount of a compound that impedes disruption of intact DMS by: (i) reducing the mean tissue volume of disrupted DMS; (ii) reducing the ratio of the number of inflammatory cells per DMS; or (iii) increasing the ratio of the number of macrophages containing disrupted DMS material per DMS, each when compared to controls. The compound impedes the dirsuption of intact DMS when administered, at an in-tissue concentration of about 10xe2x88x925 M or less, to an experimental animal or to a test preparation of human postmortem brain that has received an intracerebral injection of DMS. In one preferred embodiment, the compound thus administered impedes disruption of intact DMS by reducing the mean tissue volume of disrupted DMS material. The compound acts on DMS components in such a way that disrupted DMS in situ attain a significantly reduced diameter, volume of tissue occupied by the redistributed transformed DMS protein material, and associated injury foci, when compared to the diameter, volume of tissue occupied, and injury foci of disrupted DMS in an untreated subject or in a subject treated with an inactive agent.
In accordance with another aspect of the present invention, there is provided a composition for treating cerebral amyloidosis, comprising a pharmaceutically effective amount of a compound that impedes disruption of intact DMS by: (i) reducing the mean tissue volume of disrupted DMS; (ii) reducing the ratio of the number of inflammatory cells per DMS; or (iii) increasing the ratio of the number of macrophages containing disrupted DMS material per DMS, each when compared to controls. The compound impedes the dirsuption of intact DMS when administered, at an in-tissue concentration of about 10xe2x88x925 M or less, to an experimental animal or to a test preparation of human postmortem brain that has received an intracerebral injection of DMS. The compositions and methods of using the compositions typically include at least one of the aforementioned compounds in combination with a pharmaceutically acceptable sterile vehicle, as described in REMINGTON""S PHARMACEUTICAL SCIENCES: DRUG RECEPTORS AND RECEPTOR THEORY, (18th ed.), Mack Publishing Co., Easton Pa. (1990).
Compounds also are provided that are effective in reducing the number of subsequent DMS disruptions brought about by an initial DMS disruption by reducing the ratio of the number of inflammatory cells per DMS, i.e., reducing the extent of the injury foci associated with the transformation and redistribution of protein material of an initial disrupted DMS. These compounds, when administered to a test animal that has received an injection of DMS at an in-tissue concentration of about 10xe2x88x925 M or less, result in less evidence of inflammatory reaction in relation to persistent DMS materials at the DMS injection site measured, for example, by the number of inflammatory cells such as polymorphonuclear leukocytes and mononuclear leukocytes per DMS in each injury focus, or the tissue volume taken up by non-macrophage inflammatory cells per DMS in each injury focus, when compared to controls at the same time intervals, for instance, at 24 hours and 48 hours, respectively.
In accordance with yet another aspect of the present invention, compounds are provided that are effective in reducing the number of subsequent DMS disruptions brought about by an initial DMS disruption by increasing the ratio of the number of macrophages containing disrupted DMS material per DMS, i.e., promoting the digestion and removal of the DMS material through the reticuloendothelial system. By promoting the digestion and removal of the DMS material, the compounds reduce the extent and duration of the effect of the disrupted DMS and therefore impede subsequent DMS disruptions. These compounds, when administered to a test animal that has received an injection of DMS at an in-tissue concentration of about 10xe2x88x925 M or less, result in more evidence of digestion and removal by the reticuloendothelial system, when compared to controls. Digestion and removal can be measured, for example, by the presence of intracellular intact DMS protein material within the cytoplasm of macrophages and mononuclear phagocytes and the numbers thereof at or near the site of DMS disruption, or the presence of intracellular digested, altered, proteolyzed or otherwise transformed DMS protein material within the cytoplasm of macrophages and mononuclear phagocytes and the numbers thereof at or near the site of DMS disruption.
Illustrative of the compounds within the present invention include those of general formula (A): 
where X is selected from 
and Y is selected from 
In the general formula (A) above:
R1 and R2 are each one or more independent substituents selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C3-C5 cycloalkyl, C1-C5 alkoxy, C2-C5 alkynyl, halogen, C1-C5 alkoxy, C1-C5 haloalkyl, alkylamino, phenyl, nitro, carboxyl, piperazinyl, pyridyl, and fused ring systems; wherein if R1 or R2 are piperazinyl, a nitrogen atom on each piperazinyl may be substituted with a moiety selected from C1-C5 alkyl, C3-C5 cycloalkyl, C1-C5 haloalkyl, substituted or unsubstituted C1-C5 alkylamino; wherein if R1 or R2 are alkylamino, each alkylamino consists of 1 to 5 carbon atoms and the amino group is unsubstituted piperazinyl, a nitrogen atom on each piperazinyl may be substituted with a moiety selected from C1-C5 alkyl, C3-C5 cycloalkyl, C1-C5 haloalkyl, substituted or unsubstituted C1-C5 alkylamino; wherein if R1 or R2 are alkylamino, each alkylamino consists of 1 to 5 carbon atoms and the amino group is unsubstituted or mono- or di-substituted with C3-C5 cycloalkyl, C2-C5 alkenyl, C2-C5 alkynyl or C1-C5 alkyl, and if R1 or R2 is a fused ring system, each individual R1 or R2 involved in the fused ring system, together with the phenyl to which it is attached, forms a fused ring system selected from naphthalene, anthracene, acenaphthylene, fluorene, phenalene, phenanthrene, fluoranthene, acephenanthrylene, aceanthrylene, aceanthrylene, isoindole, indole, quinolizine, isoquinoline, phthalazine, quinoxaline, quinoline, phthalazine, quinazoline, and cinnoline, wherein one or more carbon atoms in the fused ring may be replaced with a nitrogen atom and each ring may be saturated or unsaturated, in whole or in part; wherein each ring may be substituted by one or more substituents selected from halogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, and C1-C5 haloalkyl;
R3 is hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C3-C5 cycloalkyl, C2-C5 alkynyl, amino, C1-C5 alkyl-substituted amino, sulfur, oxygen, phenyl, benzyl, naphthyl and anthracenyl; wherein each aromatic ring may be substituted by one or more substituents selected from halogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkylyl, and C1-C5 haloalkyl;
R4 is hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C3-C5 cycloalkyl, C2-C5 alqynyl, amino, C1-C5 alkyl-substituted amino, C1-C5 alkylamino, C2-C5 alkenylamino, C1-C5 alkyl substituted C1-C5 alkylamino, C1-C5 alkyl substituted C2-C5 alkenylamino, sulfur, oxygen, phenyl, benzyl, naphthyl and anthracenyl; wherein each aromatic ring may be substituted by one or more substituents selected from halogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, and C1-C5 haloalkyl;
R5 is nitrogen or carbon;
R6 and R7 are each one or more independent substitutions selected from hydrogen, C1-C5 alkyl, C2-C5 alkenyl, C3-C5 cycloalkyl, C1-C5 alkoxy, C2-C5 alkynyl, halogen, C1-C5 haloalkyl, alkylamino, phenyl, nitro, carboxyl, piperazinyl, pyridyl, and fused ring systems; wherein if R6 or R7 is piperazinyl, a nitrogen atom on each piperazinyl may be substituted with a moiety selected from C1-C5 alkyl, C3-C5 cycloalkyl, C1-C5 haloalkyl, substituted or unsubstituted C1-C5 alkylamino; wherein if R6 or R7 is alkylamino, each alkylamino consists of 1 to 5 carbon atoms and the amino group is unsubstituted or mono- or di-substituted with C3-C5 cycloalkyl, C2-C5 alkenyl, C2-C5 alkynyl or C1-C5 alkyl, and if R6 or R7 is a fused ring system, each individual R6 and R7 involved in the fused ring system, together with the phenyl to which it is attached, forms a fused ring system selected from naphthalene, anthracene, acenaphthylene, fluorene, phenalene, phenanthrene, fluoranthene, acephenanthrylene, aceanthrylene, aceanthrylene, isoindole, indole, quinolizine, isoquinoline, phthalazine, quinoxaline, quinoline, phthalazine, quinazoline, and cinnoline, wherein one or more carbon atoms in the fused ring may be replaced with a nitrogen atom and each ring may be saturated or unsaturated, in whole or in part; wherein each ring may be substituted by one or more substituents selected from halogen, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, and C1-C5 haloalkyl;
n is an integer of from 0 to 5;
m is an integer of from 0 to 5;
or a pharmaceutically acceptable salt of such compound.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Unless otherwise specified, the respective contents of documents cited in the following description are hereby incorporated by reference.